GSK-3β controls autophagy by modulating LKB1-AMPK pathway in prostate cancer cells

Prostate. 2016 Feb;76(2):172-83. doi: 10.1002/pros.23106. Epub 2015 Oct 6.

Abstract

Background: Glycogen synthase kinase 3β (GSK3B, GSK-3β) is a multi-functional protein kinase involved in various cellular processes and its activity elevates after serum deprivation. We have shown that inhibition of GSK-3β activity triggered a profound autophagic response and subsequent necrotic cell death after serum deprivation in prostate cancer cells. In this study, we dissected the mechanisms involved in GSK-3β inhibition-triggered autophagy.

Methods: Prostate cancer PC-3 and DU145 cells were used in the study. Multiple GSK-3β specific inhibitors were used including small chemicals TDZD8, Tideglusib, TWS119, and peptide L803-mts. Western blot assay coupled with phospho-specific antibodies were used in detecting signal pathway activation. ATP levels were assessed with ATPLite kit and HPLC methods. Autophagy response was determined by evaluating Microtubule-associated proteins 1A/1B light chain 3B (LC3B) processing and p62 protein stability in Western blot assays. Immunofluorescent microscopy was used to detect LKB1 translocation.

Results: Inhibition of GSK-3β activity resulted in a significant decline of cellular ATP production, leading to a significant increase of AMP/ATP ratio, a strong trigger of AMP-activated protein kinase (AMPK) activation in prostate cancer PC-3 cells. In parallel with increased LC-3B biosynthesis and p62 protein reduction, the classical sign of autophagy induction, AMPK was activated after inhibition of GSK-3β activity. Further analysis revealed that Liver kinase B1 (LKB1) but not Calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) is involved in AMPK activation and autophagy induction triggered by GSK-3β inhibition. Meanwhile, GSK-3β inhibition promoted LKB1 translocation from nuclear to cytoplasmic compartment and enhanced LKB1 interaction with its regulatory partners Mouse protein-25 (MO25) and STE20-related adaptor (STRAD).

Conclusions: In conclusion, our data suggest that GSK-3β plays an important role in controlling autophagy induction by modulating the activation of LKB1-AMPK pathway after serum deprivation.

Keywords: AMPK; GSK-3β; LKB1; autophagy; serum deprivation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinase Kinases
  • AMP-Activated Protein Kinases / physiology*
  • Animals
  • Autophagy / drug effects
  • Autophagy / physiology*
  • Cell Line, Tumor
  • Enzyme Inhibitors / pharmacology
  • Glycogen Synthase Kinase 3 / antagonists & inhibitors
  • Glycogen Synthase Kinase 3 / physiology*
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Male
  • Mice
  • Prostatic Neoplasms / metabolism*
  • Prostatic Neoplasms / pathology
  • Protein Serine-Threonine Kinases / physiology*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*

Substances

  • Enzyme Inhibitors
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, mouse
  • Protein Serine-Threonine Kinases
  • STK11 protein, human
  • Glycogen Synthase Kinase 3
  • AMP-Activated Protein Kinase Kinases
  • AMP-Activated Protein Kinases